Intramolecular dynamics and structural determinants regulating the in vivo and in vitro functions of a bi-directional kinesin motor

Mitosis is a conserved process by which duplicated chromosomes are transferred from mother to daughter cells, by the microtubule (MT)-based mitotic spindle. One of the major factors that govern the mitotic spindle dynamics are the bipolar kinesin-5 motors that separate the spindle-poles apart by plus-end directed motility on spindle MTs. Interestingly, recent studies have indicated that some kinesin-5 motors can move in both plus- and minus-end direction of the MTs. Two modes of motility have been reported for such bi-directional motility: (a) context-dependent directionality reversal, in which motors undergo minus-end directed motility at the single-molecule level and switch to plus-end directed motility under different conditions, such as during MT gliding or antiparallel sliding or as a function of motor clustering; and (b) bi-directional motility, defined as movement in two directions in the same assay, without persistent unidirectional motility. We examine how modulation of motor-MT interactions affects these two modes of motility for the bi-directional kinesin-5, Cin8. We report that the large insert in loop-8 (L8) within the motor domain increases the MT affinity of Cin8 in vivo and in vitro and is required for Cin8 intracellular functions. We found that L8 induces single Cin8 motors to behave according to context-dependent directionality reversal and bi-directional motility modes at intermediate ionic strength and according to a bi-directional motility mode in an MT surface-gliding assay under low motor density conditions. We propose that L8 facilitates flexible anchoring of Cin8 to the MTs, which enables the direct observation of bi-directional motility in motility assays.